The underlying goal of this research program is to understand the mechanism of regulation of biological activity in macromolecular complexes through structural analysis. We use X-ray crystallography as a tool to achieve this objective. 1. Serotonin N-acetyltransferase and 14-3-3. Serotonin N-acetyltransferase (or arylalkylamine N-acetyltransferase, AANAT) controls the circadian variation of circulating melatonin levels in mammals. Preliminary insight into the regulation of AANAT is provided by the result that AANAT is phosphorylated in pineal cells, and that one of the phosphorylation sites is contained within a characteristic binding motif for the 14-3-3 class of cytoplasmic adaptor molecules. Biological roles of 14-3-3 complexes have been demonstrated in signal transduction, subcellular targeting, and cell cycle control. During the past year, we have crystallized and determined the structure of phosphorylated AANAT and 14-3-3 zeta. The stable 2:2 complex illustrates how 14-3-3 scaffolding proteins can modulate enzyme activity. The complex consists of a 14-3-3 dimer and two well-defined AANAT molecules. The structure supports the notion that 14-3-3 binding stabilizes the catalytically relevant conformational state of AANAT in which the AcCoA binding pocket is already open and poised for substrate binding. To confirm the relevance of this crystallographic observation, we performed isothermal calorimetry (ITC) measurements to determine the effect of 14-3-3 binding on the dissociation constants of AANAT's substrates. These revealed that the binding of both serotonin and AcCoA is significantly tighter to the 14-3-3/AANAT complex than to AANAT alone. Activity measurements performed at physiological substrate concentrations showed an increase in activity of the complex relative to AANAT alone, suggesting that phosphorylation-induced protein-protein complex formation might contribute to the regulation of melatonin production in pineal cells. 2. Adeno-associated virus (AAV) Rep mediated site-specific chromosomal integration. AAV has the unique ability to site-specifically integrate its genome into human chromosome 19, a property which places it in the forefront of much current interest as a vector for gene therapy. The viral DNA encodes for the Rep protein which possesses a site-specific DNA binding activity, an endonuclease activity, an ATPase activity, and a 3'-to-5' helicase activity. The structural basis of these activities is currently unknown. To shed light onto the mechanism of integration, we have expressed and purified several constructs encoding single and multiple domains of AAV Rep. We have recently obtained crystals of the N-terminal catalytic fragment, and are in the process of improving their resolution. We have also prepared a series of DNA substrates that span the viral terminal resolution site, and are currently characterizing the Rep complexes that assemble on these substrates.